AU2020430369B2

AU2020430369B2 - Thieno[2,3-d]pyrimidine HIV-1 non-nucleoside reverse transcriptase inhibitor, preparation method therefor and use thereof

Info

Publication number: AU2020430369B2
Application number: AU2020430369A
Authority: AU
Inventors: Dongwei KANG; Xinyong LIU; Peng ZHAN
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-02-21
Filing date: 2020-03-03
Publication date: 2023-04-13
Anticipated expiration: 2040-03-03
Also published as: AU2020430369A1; WO2021164052A1; CN111217833B; CN111217833A; US20230067045A1

Abstract

Disclosed are a thieno[2,3-d]pyrimidine HIV-1 reverse transcriptase inhibitor, DK6-1, a preparation method therefor and the use thereof, a pharmaceutical composition containing DK6-1 and the use of the compound and a composition containing one or more such compounds in the preparation of a drug for treating and preventing human immunodeficiency virus (HIV).

Description

Thiophene[2,3-dlpyrimidine derivative DK6-1 and its preparation method and application

Field of the invention

The present invention relates to the field of organic compound synthesis and pharmaceutical

applications, and more particularly to thiophene[2,3-d]pyrimidine derivative DK6-1 and its

preparation method and as an application of HIV-1 inhibitor.

Background of the invention Acquired Immune Deficiency Syndrome (AIDS) has become a major infectious disease endangering human life and health at present, and its main pathogen is human immunodeficiency virus type 1 (HIV-1). Although the implementation of highly active antiretroviral therapy (HAART) significantly prolonged the survival of patients, the problems of drug resistance, serious side effects and long-term drug costs forcing researchers to develop novel HIV-1 inhibitors with higher potency and lower toxicity. HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) have gained an increasingly important role in highly active antiretroviral therapy (HAART) regimens used to treat AIDS patients for their potent antiviral activity, high selectivity, and lack of mitochondrial toxicity which characterizes the NRTIs. However, drug-resistant mutants rapidly emerge with their clinical applications because of their allosteric mechanism of action and low genetic barrier. Therefore, the development of novel high-efficiency and low-toxicity NNRTIs has always been one of the hot spots of anti-HIV drug research. Diarylpyrimidine (DAPY) is a typical class of HIV-1 NNRTIs, which has effective activity against wild-type and mutant HIV-1 strains. Up to now, two DAPY NNRTIs etravirine (ETV) and rilpivirine (RPV) have been approved by the U.S. FDA. However, these compounds have poor water solubility and led to their low oral bioavailability. With ETR and RPV as the lead compounds, our previous efforts have led to the design and synthesis of two novel NNRTIs K-5a2 and 25a. However, both compounds suffered from a stronger human ether-A-go-go related gene (hERG) inhibitory activity (K-5a2, IC5 o = 0.130 pM; 25a, IC5 o = 0.186 pM). In addition, 25a showed higher cytotoxicity (CC 5 o = 2.30 pM) and lower bioavailability (F= 16.19%). Therefore, further structure modification is still needed to achieve improved antiviral potency, decreased toxicity, and favorable pharmacokinetic properties.

0 H2 ONH 2

CN ON ON CNN ON ON NH N N N NH ON NH

HN N NH N Br NN S N NH 2 Etravirine Rilpivirine K-5a2 25a

Summary of the invention

The present invention provides a thiophene[2,3-d]pyrimidine derivative DK6-1 and a

preparation method thereof. The invention also provides the use of thiophene[2,3-d]pyrimidine

derivative DK6-1 as HIV-1 inhibitor.

The technical proposal of the invention is as follows:

1, Thiophene[2,3-d]pyrimidine derivative DK6-1 The invention provides thiophene[2,3-d]pyrimidine derivative DK6-1, and pharmaceutically acceptable salt, ester or prodrug thereof. CN / S0 2 NH 2

N

o NYNH

CIN

DK6-1

2. Preparation of thiophene[2,3-dlpyrimidine derivative DK6-1

The preparation method of thiophene[2,3-d]pyrimidine derivative DK6-1 is as follows: the commercially available 2,4-dichlorothiophene[2,3-d]pyrimidine (1) was selected as starting

material, which was treated with 3,5-dimethyl-4-hydroxybenzaldehyde afforded intermediate 2.

The cyanovinyl compound 3 was obtained by reaction of 2 with diethyl cyanomethylphosphonate under Wittig-Homer reaction. Then 3 was treated with N-(tert-butoxycarbonyl)-4-aminopiperidine

and trifluoroacetic acid to yield the key intermediate 4, which was reacted with

4-(bromomethyl)benzenesulfonamide to give the target compound DK6-1.

CN CN CN / SO 2 NH 2

CHO H

C1 N CI iv N 1 0 N CI 0 N YC 0 N NH 0 N NH S N N N N S S S 1 2 3 4 DK6-1

Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzaldehyde, DMF, K 2 C0 3 , 20-30°C; (ii)

(EtO) 2 P(O)CH 2 CN, t-BuOK, THF/DCM, 0°C; (iii) 4-(tert-butoxycarbonyl)aminopiperidine, DMF,

K 2 C0 3 , reflux; then TFA, DCM, 20-30°C; (iv) 4-(bromomethyl)benzenesulfonamide, DMF, K2 C0 3

, -30 0 C.

3. Activity evaluation and application of thiophene[2,3-dpyrimidine derivative DK6-1

Antiviral potency was evaluated in MT-4 cell cultures infected with WT HIV-1 strain (I1IB) as well as cells infected with a panel of NNRTI-resistant single- and double-mutant strains, such as

L1001, K103N, Y181C, Y188L, E138K, F227L+V106A and K103N+Y181C (RES056). Etravirine

(ETR) was selected as control drug. The values of EC5 o (anti-HIV activity) and CC 5 o (cytotoxicity) was depicted in Figure 1.

DK6-1 showed potent activity against wild-type and mutant HIV-1 strains, being superior to that of

ETR. For HIV-1 wild-type and K103N mutant strains, DK6-1 exhibited an EC5 o values of 3.24 nM and 2.34 nM, being comparable to that of ETR; for L100I, Y181C, Y188L and E138K, DK6-1 was

demonstrated with EC 5 ovalues less than 8 nM, being 2-fold more potent than that of ETR; in the

case of F227L+V106A and RES056, the activity of DK6-1 was 6-fold and 3-fold potent than that of ETR, respectively. Moreover, the cytotoxicity of DK6-1 (CC5 o = 10.1 pM) was much decreased

compared to that of 25a (CC5 o = 2.30 pM).

Then, MT-4 cells infected with HIV-1 IIB was passaged for 30 times in the presence of DK6-1. Several mutations were detected in the RT gene in comparison with the DNA sequence of

the WT HIV-1 (IIB) strain, including K101E, V1081, F227C, and M2301. The activities of DK6-1

against these mutant strains sharply decreased compared to its activity against HIV-1 IIB, but he NRTIs AZT exhibited significantly higher potency against these mutant strains (EC5 o = 1.0 nM), being about 10-fold potent than its activity against HIV-1 IIIB (EC5 o = 12.7 nM). The distinct

antiviral resistance profiles of the novel discovered NNRTIs DK6-1 and the approved NRTIs AZT support the use of them together in HAART and should be helpful in the development of next generation of anti-HIV therapy with an increased genetic barrier to resistance. The pharmacokinetics study result (Figure 1) demonstrated that DK6-1 have a favorable bioavailability (F= 37.06 %) and better safety profiles (LD5 o > 2000 mg/kg). The hERG inhibitory activity result indicated that DK6-1 exhibited much reduced QT liability and lower hERG inhibition (IC5 0 = 0.98 pM) in comparison with that of 25a (IC5 0 = 0.186 pM) and K-5a2 (IC5 0 =

0.130 pM). The promising in vitro and in vivo results highlights that DK6-1 has enormous potential as a next generation anti-HIV-1 drug candidate. Also described here are thiophene[2,3-d]pyrimidine derivative DK6-1 used as HIV-1 NNRTIs,

furthermore, these HIV-1 inhibitors will be used as anti-AIDS drugs.

Also described here are pharmaceutical composition comprising thiophene[2,3-d]pyrimidine

derivative DK6-1, and with one or more kind of pharmaceutically acceptable carrier or excipient

The present invention provides a novel thiophene[2,3-d]pyrimidine derivative DK6-1, its

preparation method, anti-HIV-1 activity screening result and its first application in the field of

antiviral. The thiophene[2,3-d]pyrimidine derivative DK6-1 of the present invention have been

proved to be useful as HIV-1 inhibitor and have high application value. In particular, it can be used

as anti-AIDS drug.

Unless the context clearly requires otherwise, throughout the description and the claims,

the words "comprise", "comprising", and the like are to be construed in an inclusive sense as

opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not

limited to".

Figures and figure legends

Figure 1 shows the comparison of cell activity and cytotoxicity between ETR and DK6-1. Figure 2 shows the comparison of hERG inhibitory activity between 25a and DK6-1.

Examples

Selected examples are listed as follows, the invention includes these compounds disclosed herein but not confined to them.

Example 1: The preparation of (E)-3-(3,5-dimethyl-4-((2-(piperidin-4-ylamino)thieno[2,3-dlpyrimidin-4-yl)oxy)phenyl)acrylo nitrile (4) CN

H N O N NH

4

A mixture of 4-hydroxy-3,5-dimethylbenzaldehyde (1.76 g, 11.7 mmol) and K 2C03 (2.70 g,

19.5 mmol) in 40 mL of DMF was stirred at room temperature for 15 min, and then

2,4-dichlorothiopheno[2,3-d]pyrimidine (1, 2.0 g, 9.76 mmol) was added to the mixture. The

mixture was stirred for another 1.5 h and then poured into ice water (200 mL) and left to stand for

min. The obtained precipitated was filtrated and washed with cold water, recrystallized from

DMF-H 20 to provide intermediate 2 as a white solid in 85% yield, mp: 263-265°C. ESI-MS: mlz

319.4 (M + 1), 341.2 (M + Na). 1C5 H1 iiClN 2 0 2S (318.02).

A mixture of (EtO) 2P(O)CH 2CN (1.34 g, 7.52 mmol) and t-BuOK (1.42 g, 12.5 mmol) in THF

(25 mL) was stirred for 1 h at 0 °C, and then a solution of 2(2.0 g, 6.28 mmol) in THF (15 mL) and

DCM (15 mL) was slowly added over 1 h. The mixture was stirred for another 4 hours at room

temperature and then poured into ice water (60 mL). The precipitate was collected and washed with

water to give intermediate 3 as a white solid in 72% yield, mp: 235-237°C. ESI-MS: mlz 342.4 (M

+ 1), 364.2 (M + Na). C 17H 12 ClN 3 0S (341.04).

Compound 3 (0.34 g, 1.0 mmol), N-Boc-4-aminopiperidine (0.24 g, 1.2 mmol), and anhydrous

K 2C3 (0.28 g, 2 mmol) were added in DMF (10 mL) and refluxing 8 h under magnetic stirring

(monitored by TLC). Then the mixed solution was cooled to room temperature and 50 mL of ice

water was added. The resulting precipitate was collected and dissolved in DCM (5 mL) and

trifluoroacetic acid (TFA) (0.74 mL, 10 mmol). The mixed solution was stirred for another 3 h

(monitored by TLC) at room temperature. Then the reaction solution was alkalized to pH 9 with

saturated sodium bicarbonate solution and washed with saturated sodium chloride solution (10 mL).

The aqueous phase was extracted with DCM (3 x 5 mL). Then the combined organic phase was

dried over anhydrous Na2SO 4, filtered, and concentrated under reduced pressure to give 4 as a

white solid in 71% yield, mp 123-125°C. ESI-MS: m/z 406.3 (M +1). C 22 H 2 3N5 0S (405.16).

Example 2: The preparation of DK6-1 Compound 4 (0.20 g, 0.5 mmol) and anhydrous K 2 CO3 (0.14 g, 1.0 mmol) were added to

anhydrous DMF (10 mL), which was followed by addition of 4-(bromomethyl)benzenesulfonamide

(0.15 g, 0.6 mmol). The reaction mixture was stirred at room temperature for 6 h (monitored by

TLC). Then the solvent was removed under reduced pressure, and water (30 mL) was added,

extracted with ethyl acetate (3 x 10 mL), and the organic phase was washed with saturated sodium

chloride (10 mL), then dried over anhydrous Na2SO4 to give the corresponding crude product,

which was purified by flash column chromatography and recrystallized from ethyl acetate

(EA)/petroleum ether (PE) to afford the target compound DK6-1. White solid in 67% yield, mp

205-207 0C. 'H NMR (400 MHz, DMSO-d) 6 7.78 (d, J= 8.2 Hz, 2H, C 3,C 5-Ph'-H), 7.61 (d, J=

13.6 Hz, 1H, ArCH=), 7.48-7.45 (m, 4H), 7.35 (d, J= 5.9 Hz, 1H, C7-thienopyrimidine-H), 7.31 (s,

2H, SO 2 NH2 ), 7.25 (d, J= 6.0 Hz, 1H, C6-thienopyrimidine-H), 7.07 (s, 1H, NH), 6.43 (d, J= 16.7

Hz, 1H, =CHCN), 3.72-3.70 (m, 1H), 3.49 (s, 2H, N-CH 2), 2.74-2.72 (m, 2H), 2.08 (s, 6H), 13 1.99-1.27 (m, 6H). C NMR (100 MHz, DMSO-d) 6 162.6, 159.3, 150.5, 143.4, 143.1, 131.7,

131.7, 129.4, 128.6, 126.0, 119.4, 118.9, 96.7, 62.0, 60.2, 52.7, 31.6, 21.1, 16.6, 14.5. ESI-MS: m/z

575.3 (M + 1), 597.5 (M + Na). C 2 9 H3 N 60 3 S 2 (574.18).

Example 3. In vitro anti-HIV activity of DK6-1 Selected compounds were screened for inhibitory activity against HIV-1 using MTT method

as describe previously by Christophe. Pannecouque et al. Nat. Protoc. 3 (2008) 427-434, and Rudi

Pauwels et al. J Virol. Methods 20(1988) 309-321. And in vitro anti-HIV activity of compounds

were supported by Rega Institute for Medical Research. The MTT assay is based on the reduction

of the yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by

mitochondrial dehydrogenases of metabolically active cells to a blue formazan which can be

measured spectrophotometrically. Tested optical density served as an indicator for live cells, and

survival rate can be concluded by testing the optical density of 540 nm and 690 nm. MT-4 cells

infected with HIV-1 can only survive for 5 to 7 days without any treatment, but when HIV-1

inhibitors were added, they can protect MT-4 cell from cytopathic. Serial solution of compounds

was added to MT-4 cells after infected with HIV-1, MTT method was used to detect the survival

rate after culture for 5 to 7 days. EC 5 o value was defined as compound concentration required to achieve 50% protection of MT-4 cells against HIV-1-induced cytopathic effect. Materials

(1) MT-4 cells infected with HIV-1 viral strains (IIB, K103N, Y181C, Y88L, Y181C/K103N)

were provided by Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium.

(2) MTT and formazan: sigma Chemical Co.

(3) Preparation of compounds: Stock solutions (10 x final concentration) of test compounds is

diluted with double distilled H 2 0 for 5 folds and 5 concentrations of one compound are prepared.

(4) Reference drug: Etravirine (ETR).

(5) Test method (MTT method): Serial five-fold dilutions of test compounds were added to

cultured MT-4 cells infected with HIV-1, after 5 to 7 days, MTT was added and cultured for a few

hours. Medium was removed and lysate was added followed by formazan, OD value was

determined in 690 nm and 540nm by microplate reader, and EC5 o value was calculated. Methods

The MTT method was described briefly as follows: 96-well plastic microtiter trays were filled

with 100 L of complete medium. Subsequently, serial of tested compounds was added (25 L) to

two series of triplicate wells so as to allow simultaneous evaluation of their effects on HIV- and

mock infected cells. 50 L of 1 x 104 cells/mL MT-4 cells were added. After cultured for 5 days at

37°C in humidified atmosphere in the presence of 5% C02, MTT was added and cultured for

another 2h, then medium was removed and 100 L isopropanol solution was added to lyse the cells.

Formazan crystals were added and vibrated platform shaker for 10 min to solubilize the formazan

crystals. Absorbances at 690 nm and 540 nm were read by using spectrophotometrically. EC5 o was

defined as the concentration achieving 50% protection from the cytopathic effect of the virus in

infected cells. The results are shown in Figure 1.

Example 4. Pharmacokinetics assays of DK6-1

Ten male Wistar rats (180-200 g) were randomly divided into two groups to receive

intravenous (2 mg.kg-1) and oral administration (20 mg.kg-1) of the compounds. A solution of DK6-1 was prepared by dissolving in polyethylene glycol (peg) 400/normal saline (65/35, V/V).

Blood samples of the intravenous group were collected from the jugular sinus at 2 min, 5 min, 15

min, 30 min, 1 h, 1.5 h, 2 h, 4 h, and 8 h after dosing, and blood samples of the oral administration group were collected at 5 min, 15 min, 30 min, 1h, 2 h, 4 h, 6 h, 8h, 10 h, and 12 h after dosing

(200 pL of blood each times). All the samples were then centrifuged at 8000 rpm for 8 min to separate plasma. The concentration of DK6-1 in plasma was determined by LC-MS/MS analysis.

Briefly, 50 pL of plasma was added to 50 L of internal standard and 300 pL of methanol in a 5 mL

centrifugation tube, which was centrifuged at 3000g for 10 min. The supernatant layer was collected and a 20 pL aliquot was injected for LC-MS/MS analysis. Standard curves for DK6-1 in

blood were generated by the addition of various concentrations of DK6-1 together with internal

standard to blank plasma. Then all samples were quantified with an Agilent 1200 LC/MSD (Agilent, USA). The mobile phase was methanol/1.5% glacial acetic acid (60:40, V/V) at a flow

rate of 1.0 mL/min. The plasma pharmacokinetic data were analyzed by using the non-av model of DAS 2.0

pharmacokinetic program. The main pharmacokinetic parameters (Cmax, AUC, Tmax, Ti/ 2 , and CL)

were calculated. The results are shown in Table 1.

Table 1. Pharmacokinetics evaluation of DK6-1 T1/2 Tinax Cnax AUCo-t AUCo-- CL F Subject (h) (h) (ng/mL) (h*ng/mL) (h*ng/mL) (L/h/kg) (%)

DK6-1 (iv) 2.0±0.4 0.033 1713±399 814±179 887±174 2.3±0.4

DK6-1 (po) 2.8±0.3 3.2±0.9 614±249 3017±547 3287±517 - 37.06

Example 5: Assay procedures for hERG activity The inhibitory activity against the hERG potassium channel was tested in HEK293 cells

which were stably transfected with hERG cDNA 2 6 . HEK239 cells expressing hERG were cultured

in 35 mm dishes for 24 hours and kept at 37°C under 5% C02. A micropipette was drawn out from

borosilicate glass to give a tip resistance between 3 ~ 5 MQ. For each trial, one dish of cells was

removed from the incubator, washed twice and placed on the microscope. The whole-cell

recordings were performed using a commercial patch clamp amplifier.

Tail currents were evoked once every 30 s by a 3 s, -50 mV repolarizing pulse following a 2 s,

+50 mV depolarizing pulse with a stable voltage of -80 mV. The voltage protocol started with a

ms depolarization pulse of -50mv, which served as the baseline for calculating the peak tail

current amplitude. Only stable cells with recording parameters exceeding the threshold were used

in the experiments. The hERG current was allowed to stabilize for 3 minutes. The cells were kept

in the test solution until the peak tail current was stable (< 5% change) for -5 sweeps. Peak tail amplitudes were then plotted as a function of the sweep number. Before testing the composite application, the average of the five peak tail currents in the steady state was taken as the control current amplitude. Four or five peak tail current measurements at the steady state after test compound application were averaged as the residual current amplitude after the test compound was suppressed. The result is shown in Figure 2.

Claims

Claim What is claimed is:

1. A compound of formula DK6-1 or a pharmaceutically acceptable salt, ester or prodrug

thereof

CN / SO 2NH 2

N

O N NH

N

S DK6-1

2. The compound of claim 1 or a pharmaceutically acceptable salt or prodrug thereof wherein

the pharmaceutically acceptable salt is hydrochloride, sulfate, tartrate or citrate.

3. The compound of claim 1 having a melting point of 205-207°C.

4. The compound claim 1 having the following spectral data: 'H NMR (400 MHz, DMSO-d6

) 6 7.78 (d, J= 8.2 Hz, 2H, C 3,C-Ph'-H), 7.61 (d, J= 13.6 Hz, 1H, ArCH=), 7.48-7.45 (m, 4H), 7.35 (d, J= 5.9 Hz, 1H, C7-thienopyrimidine-H), 7.31 (s, 2H, SO2 NH 2 ), 7.25 (d, J= 6.0 Hz, 1H,

C6-thienopyrimidine-H), 7.07 (s, 1H, NH), 6.43 (d, J= 16.7 Hz, 1H, =CHCN), 3.72-3.70 (m,1H), 3.49 (s, 2H, N-CH 2), 2.74-2.72 (m, 2H), 2.08 (s, 6H), 1.99-1.27 (m, 6H); 1 3 C NMR (100 MHz, DMSO-d) 6 162.6, 159.3, 150.5, 143.4, 143.1, 131.7, 131.7, 129.4, 128.6, 126.0, 119.4, 118.9,

96.7, 62.0, 60.2, 52.7, 31.6, 21.1, 16.6, 14.5. ESI-MS: m/z 575.3 (M + 1), 597.5 (M + Na).

C 29H 3 0N 6 03 S2 (574.18).

5. A method of preparing the compound of claim 1 comprising the following steps:

CN CN CN / SO 2 NH2

CHO H NN

CI N CN - iv 1 N N CI 0 NYC 0 N NH 0 N NH S N N NN

1 2 3 4 DK6-1

wherein i) is reaction of1 with 3,5-dimethyl-4-hydroxybenzaldehyde to afford 2; ii) is reaction of 2

with diethyl cyanomethylphosphonate under Wittig-Horner reaction conditions to afford 3; iii) is

reaction of 3 with N-(tert-butoxycarbonyl)-4-aminopiperidine in trifluoroacetic acid to afford 4 and

iv) is reaction of 4 with 4-(bromomethyl)benzenesulfonamide to afford a compound of formula

DK6-1.

6. The method of claim 5 wherein each respective step uses reagents and conditions as follows:

(i) 3,5-dimethyl-4-hydroxybenzaldehyde, DMF, K2 C0 3 , 20-30°C; (ii) (EtO) 2 P(O)CH 2 CN,

t-BuOK, THF/DCM, 0°C; (iii) 4-(tert-butoxycarbonyl)aminopiperidine, DMF, K 2 C0 3 , 120°C; then

TFA, DCM, 20-30°C; (iv) 4-(bromomethyl)benzenesulfonamide, DMF, K 2C0 3,20-30°C.

7. A method of treatment or prevention of HIV comprising the step of administering to a

subject in need an effective amount of a compound according to claim 1.

8. A method of treatment of HIV comprising the step of administering to a subject in need an

effective amount of a compound according to claim 1.

9. A method of prevention of HIV comprising the step of administering to a subject in need an

effective amount of a compound according to claim 1.

10. A method of treatment of AIDS comprising administering a compound of claim 1 in

combination with an NRTI as a main component of HAART therapy.

11. The use of a compound according to claim 1 in the manufacture of a medicament for the treatment or prevention of HIV

12. The use of a compound according to claim 1 in the manufacture of a medicament for the

treatment of HIV

13. The use of a compound according to claim 1 in the manufacture of a medicament for the

prevention of HIV

14. A method of prevention of HIV comprising the step of administering to a subject in need

an effective amount of a compound according to claim 1.

15. The use of a compound according to claim 1 in the manufacture of a medicament for

administration with an NRTI as a main component of HAART therapy for the treatment of AIDS.

16. A pharmaceutical composition comprising the compound of claim 1.

17. A pharmaceutical composition comprising the compound of claim 1 and a

pharmaceutically acceptable vector.

18. A pharmaceutical composition comprising the compound of in claim 1 and one or more

medically acceptable excipients.